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Mouse Dazap2 Knockout Project (CRISPR/Cas9)

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Mouse Dazap2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Dazap2 Knockout Project (CRISPR/Cas9) Objective: To create a Dazap2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Dazap2 gene (NCBI Reference Sequence: NM_011873 ; Ensembl: ENSMUSG00000000346 ) is located on Mouse chromosome 15. 4 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 4 (Transcript: ENSMUST00000000356). Exon 1~4 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a gene trap insertion are viable, fertile and do not exhibit motor coordination or balance defects. Exon 1 starts from about 0.2% of the coding region. Exon 1~4 covers 100.0% of the coding region. The size of effective KO region: ~3782 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 Legends Exon of mouse Dazap2 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of start codon is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of stop codon is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(23.05% 461) | C(25.35% 507) | T(19.15% 383) | G(32.45% 649) Note: The 2000 bp section upstream of start codon is analyzed to determine the GC content. Significant high GC-content regions are found. The gRNA site is selected outside of these high GC-content regions. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(27.15% 543) | C(22.05% 441) | T(30.2% 604) | G(20.6% 412) Note: The 2000 bp section downstream of stop codon is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr15 + 100613713 100615712 2000 browser details YourSeq 46 1643 1800 2000 96.1% chr13 + 49147794 49148037 244 browser details YourSeq 26 1626 1660 2000 89.3% chr2 - 23373973 23374006 34 browser details YourSeq 26 347 373 2000 100.0% chr1 - 65689011 65689225 215 browser details YourSeq 21 1651 1671 2000 100.0% chr5 + 77310066 77310086 21 browser details YourSeq 21 270 290 2000 100.0% chr1 + 12868443 12868463 21 browser details YourSeq 20 965 984 2000 100.0% chr1 + 99452292 99452311 20 browser details YourSeq 20 850 869 2000 100.0% chr1 + 27087715 27087734 20 Note: The 2000 bp section upstream of start codon is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr15 + 100619495 100621494 2000 browser details YourSeq 978 27 1262 2000 90.8% chr4 - 118789659 118791057 1399 browser details YourSeq 338 1 358 2000 96.7% chr13 - 12259093 12259449 357 browser details YourSeq 113 1868 2000 2000 89.9% chr9 + 35573637 35573765 129 browser details YourSeq 67 1461 1542 2000 92.6% chr16 - 29724018 29943636 219619 browser details YourSeq 66 1461 1559 2000 89.2% chr11 + 15348596 15348710 115 browser details YourSeq 65 1461 1553 2000 88.4% chr4 - 59478061 59478178 118 browser details YourSeq 64 1476 1554 2000 92.0% chr5 - 107344787 107344891 105 browser details YourSeq 59 1476 1559 2000 89.2% chr5 + 44220441 44220548 108 browser details YourSeq 56 1477 1559 2000 91.1% chr5 + 135584681 135584973 293 browser details YourSeq 54 1478 1540 2000 93.6% chr12 - 13316538 13316629 92 browser details YourSeq 53 1478 1542 2000 95.0% chr7 + 122030576 122030674 99 browser details YourSeq 52 1478 1540 2000 92.0% chr2 - 144601059 144601150 92 browser details YourSeq 50 1476 1536 2000 91.9% chr17 - 54163128 54163217 90 browser details YourSeq 49 1480 1542 2000 88.9% chr4 + 124684850 124684912 63 browser details YourSeq 48 1478 1559 2000 88.9% chr16 - 36133993 36134109 117 browser details YourSeq 47 1461 1522 2000 92.8% chr17 - 34953282 34953344 63 browser details YourSeq 47 1461 1522 2000 87.8% chr9 + 25538026 25538086 61 browser details YourSeq 47 1481 1539 2000 94.4% chr10 + 7601530 7601605 76 browser details YourSeq 45 1478 1529 2000 94.3% chr7 - 34091179 34091232 54 Note: The 2000 bp section downstream of stop codon is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Dazap2 DAZ associated protein 2 [ Mus musculus (house mouse) ] Gene ID: 23994, updated on 12-Aug-2019 Gene summary Official Symbol Dazap2 provided by MGI Official Full Name DAZ associated protein 2 provided by MGI Primary source MGI:MGI:1344344 See related Ensembl:ENSMUSG00000000346 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Brbp; Prtb; Gcap28; gt6-12; AI314727; mKIAA0058 Expression Ubiquitous expression in lung adult (RPKM 155.9), genital fat pad adult (RPKM 154.0) and 28 other tissues See more Orthologs human all Genomic context Location: 15 F1; 15 56.36 cM See Dazap2 in Genome Data Viewer Exon count: 5 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 15 NC_000081.6 (100615662..100620761) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 15 NC_000081.5 (100446093..100451192) Chromosome 15 - NC_000081.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Dazap2 ENSMUSG00000000346 Description DAZ associated protein 2 [Source:MGI Symbol;Acc:MGI:1344344] Gene Synonyms Brbp, Gcap28, Prtb, gt6-12 Location Chromosome 15: 100,615,349-100,620,761 forward strand. GRCm38:CM001008.2 About this gene This gene has 4 transcripts (splice variants), 209 orthologues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Dazap2-201 ENSMUST00000000356.9 2135 168aa ENSMUSP00000000356.8 Protein coding CCDS27841 Q9DCP9 TSL:1 GENCODE basic APPRIS P1 Dazap2-204 ENSMUST00000230661.1 751 No protein - lncRNA - - - Dazap2-202 ENSMUST00000229424.1 542 No protein - lncRNA - - - Dazap2-203 ENSMUST00000230384.1 440 No protein - lncRNA - - - Page 7 of 9 https://www.alphaknockout.com 25.41 kb Forward strand 100.61Mb 100.62Mb 100.63Mb Genes (Comprehensive set... Dazap2-201 >protein coding Dazap2-204 >lncRNA Dazap2-202 >lncRNA Dazap2-203 >lncRNA Contigs AC123724.9 > Genes < 1110013H19Rik-201lncRNA < Smagp-201protein coding (Comprehensive set... < C330013E15Rik-201lncRNA < Smagp-205protein coding < Smagp-202protein coding < Smagp-204protein coding < Smagp-203protein coding < Smagp-206protein coding Regulatory Build 100.61Mb 100.62Mb 100.63Mb Reverse strand 25.41 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding merged Ensembl/Havana Ensembl protein coding Non-Protein Coding RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000000356 5.41 kb Forward strand Dazap2-201 >protein coding ENSMUSP00000000... Low complexity (Seg) Pfam DAZ associated protein 2 PANTHER PTHR31638:SF4 DAZ associated protein 2 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 20 40 60 80 100 120 140 168 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 9 of 9.
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